Dioxygen transfer during vitamin K dependent carboxylase catalysis

Abstract

The vitamin K dependent carboxylase of liver microsomes is involved in the posttranslational modification of certain serine protease zymogens which are critical components of the blood clotting cascade. During coupled carboxylation/oxygenation this carboxylase converts glutamate residues, dihydrovitamin K, CO2, and O2 to a gamma-carboxyglutamyl (Gla) residue, vitamin K (2R,3S)-epoxide, and H2O with a stoichiometry of 1:1 for all substrates and products. In this paper we investigate the role of molecular oxygen in the reaction by following the course of the oxygen atoms using 18O2. Two different mass spectroscopic techniques, electron ionization positive ion mass spectrometry and supercritical fluid chromatography-negative ion chemical ionization mass spectrometry, were used to quantitate the amount of 18O incorporation into the various oxygens of the vitamin K epoxide product. We found that 0.95 mol atoms of oxygen were incorporated into the epoxide oxygen, 0.05 mol atoms of oxygen were incorporated into the quinone oxygen of vitamin K epoxide, and the remaining ca. 1.0 mol atoms of oxygen were incorporated into H2O. No incorporation of oxygen into vitamin K epoxide from 50% H2(18)O was observed. Thus, the carboxylase operates as a dioxygenase 5% of the time during carboxylation/oxygenation. The relevance of these findings with respect to the nonenzymic "basicity enhancement" model proposed by Ham and Dowd [(1990) J. Am. Chem. Soc. 112, 1660-1661] is discussed.

Formation and Cleavage of C–C Bonds by Enzymatic Oxidation–Reduction Reactions

“…172,173 18 O 2 incorporation studies showed one 18 O in the epoxide oxygen and 5–20% in one of the quinone oxygens. 174,175 The epoxidation mechanism proposed by Dowd et al 174 (Figure 49) is realistic when one considers the kinetics of carbonyl oxygen exchange with H 2 O. 176–178…”

Enzymatic Chemistry of Cyclopropane, Epoxide, and Aziridine Biosynthesis

“…127,140 In these unusual enzymes, the initial 1 e - reduction of O 2 (required to convert O 2 to its reactive singlet state) is believed to be carried out by the substrate which, for all known dioxygenases in this class, is a phenolic compound that can likely access a stable radical state. 140 Biochemically well characterized examples of epoxidases from this class of enzymes include the vitamin K-dependent glutamate carboxylase, an enzyme that couples the post-translational carboxylation of glutamate residues in certain proteins to the epoxidation of vitamin K, 192–195 and the dihydroxyacetanilide epoxidases, DHAE I and DHAE II, which respectively catalyze the key steps in the biosynthesis of the epoxyquinone compounds LL-C10037α ( 143 ) and MM14201 ( 144 ) (Scheme 22). The former is an antitumor agent produced by Streptomyces LL-C10037, and the latter is an anticancer compound isolated from Streptomyces MPP 3051.…”

Vitamin K and Energy Transduction: A Base Strength Amplification Mechanism

“…Following this analysis, we have carried out 1802 labeling experiments with the carboxylase in a rat liver microsomal preparation and find, in the |mie 466 0 + |mle 306| course of 12 trials, 17 + 1.4% (standard deviation) incorporation of a second atom of 180 into the product vitamin K oxide (26,31,33,34).…”